Investigation of the metabolomic crosstalk between liver sinusoidal endothelial cells and hepatocytes exposed to paracetamol using organ-on-chip technology.

Organ-on-chip technology is a promising in vitro approach recapitulating human physiology for the study of responses to drug exposure. Organ-on-chip cell cultures have paved new grounds for testing and understanding metabolic dose-responses when evaluating pharmaceutical and environmental toxicity. Here, we present a metabolomic investigation of a coculture of liver sinusoidal endothelial cells (LSECs, SK-HEP-1) with hepatocytes (HepG2/C3a) using advanced organ-on-chip technology. To reproduce the physiology of the sinusoidal barrier, LSECs were separated from hepatocytes by a membrane (culture insert integrated organ-on-chip platform). The tissues were exposed to acetaminophen (APAP), an analgesic drug widely used as a xenobiotic model in liver and HepG2/C3a studies. The differences between the SK-HEP-1, HepG2/C3a monocultures and SK-HEP-1/HepG2/C3a cocultures, treated or not with APAP, were identified from metabolomic profiles using supervised multivariate analysis. The pathway enrichment coupled with metabolite analysis of the corresponding metabolic fingerprints contributed to extracting the specificity of each type of culture and condition. In addition, we analysed the responses to APAP treatment by mapping the signatures with significant modulation of the biological processes of the SK-HEP-1 APAP, HepG2/C3a APAP and SK-HEP-1/HepG2/C3a APAP conditions. Furthermore, our model shows how the presence of the LSECs barrier and APAP first pass can modify the metabolism of HepG2/C3a. Altogether, this study demonstrates the potential of a "metabolomic-on-chip" strategy for pharmaco-metabolomic applications predicting individual response to drugs.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Investigation of steatosis profiles induced by pesticides using liver organ-on-chip model and omics analysis.

Several studies have reported a correlation between pesticides exposure and metabolic disorders. Dichlorodiphenyltrichloroethane (DDT) and permethrin (PMT), two pesticides highly prevalent in the environment, have been associated to dysregulation of liver lipids and glucose metabolisms and non-alcoholic fatty liver disease (NAFLD). However, the effects of DDT/PMT mixtures and mechanisms mediating their action remain unclear. Here, we used multi-omic to investigate the liver damage induced by DDT, PMT and their mixture in rat liver organ-on-chip. Organ-on-chip allow the reproduction of in vivo-like micro-environment. Two concentrations, 15 and 150 µM, were used to expose the hepatocytes for 24 h under perfusion. The transcriptome and metabolome analysis suggested a dose-dependent effect for all conditions, with a profile close to control for pesticides low-doses. The comparison between control and high-doses detected 266/24, 256/24 and 1349/30 genes/metabolites differentially expressed for DDT150, PMT150 and Mix150 (DDT150/PMT150). Transcriptome modulation reflected liver inflammation, steatosis, necrosis, PPAR signaling and fatty acid metabolism. The metabolome analysis highlighted common signature of three treatments including lipid and carbohydrates production, and a decrease in amino acids and krebs cycle intermediates. Our study illustrates the potential of organ-on-chip coupled to multi-omics for toxicological studies and provides new tools for chemical risk assessment.

Effects of Potassium Fertilization on Oil Palm Fruit Metabolism and Mesocarp Lipid Accumulation.

Potassium fertilization is commonly practiced in oil palm (Elaeis guineensis) plantations to increase yield. However, its effects on fruit oil content and composition are not well documented. Here, we conducted bunch, metabolomics, and oil composition analyses in two contrasting crosses (Deli × La Mé and Deli × Yangambi) grown under different K fertilization conditions. K availability impacted bunch oil content, resulting in lower water content and higher oil proportion in fruit mesocarp, in Deli × La Mé only, thus showing differential responses of crosses to K. Oil composition at maturity did not significantly change under low K conditions despite clear alterations in fruit metabolism associated with lipid production during maturation, demonstrating the resilience of oil biosynthetic metabolism. However, the analysis of variance in oil content (across K treatments and crosses) demonstrates that sugar availability, lipid synthesis rates, and metabolic recycling are all important in determining the oil content.

From Intracellular Bacteria to Differentiated Bacteroids: Transcriptome and Metabolome Analysis in Aeschynomene Nodules Using the Bradyrhizobium sp. Strain ORS285 bclA Mutant.

Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells, where they differentiate into nitrogen-fixing bacteroids. Several legume lineages force their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. strain ORS285, a symbiont of Aeschynomene spp. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells, but they cannot differentiate into enlarged polyploid and functional bacteroids. Thus, the bclA nodule bacteria constitute an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied by a first transcriptome switch involving several hundred upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving fewer genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a nonsymbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process, fueling the biogeochemical nitrogen cycle with reduced nitrogen. It also represents a promising strategy to reduce the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs, where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.

One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds.

Dormancy is an adaptive trait that blocks seed germination until the environmental conditions become favorable for subsequent vegetative plant growth. Seed dormancy is defined as the inability to germinate in favorable conditions. Dormancy is alleviated during after-ripening, a dry storage period, during which dormant (D) seeds unable to germinate become non-dormant (ND), able to germinate in a wide range of environmental conditions. The treatment of dormant seeds with ethylene (D/ET) promotes seed germination, and abscisic acid (ABA) treatment reduces non-dormant (ND/ABA) seed germination in sunflowers (Helianthus annuus). Metabolomic and transcriptomic studies have been performed during imbibition to compare germinating seeds (ND and D/ET) and low-germinating seeds (D and ND/ABA). A PCA analysis of the metabolites content showed that imbibition did not trigger a significant change during the first hours (3 and 15 h). The metabolic changes associated with germination capacity occurred at 24 h and were related to hexoses, as their content was higher in ND and D/ET and was reduced by ABA treatment. At the transcriptional level, a large number of genes were altered oppositely in germinating, compared to the low-germinating seeds. The metabolomic and transcriptomic results were integrated in the interpretation of the processes involved in germination. Our results show that ethylene treatment triggers molecular changes comparable to that of after-ripening treatment, concerning sugar metabolism and ABA signaling inhibition.

AIF loss deregulates hematopoiesis and reveals different adaptive metabolic responses in bone marrow cells and thymocytes.

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid ß-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid ß-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.

13C and 15N natural isotope abundance reflects breast cancer cell metabolism.

Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13C and 15N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes.

Can electrospray ionization help the characterization of iron gall inks? Investigation on the interactions of gallic acid with iron ions in aqueous solutions.

In the present work, an investigation has been conducted by electrospray ionization (ESI) experiments to characterize the structures of iron gall ink complexes in solution. Simple mono and polyphenolic acid molecules added to iron sulfate salts were chosen to model the recipes of ink composition. Theoretical calculations have been used (1) to determine the stability of the ionic complexes generated in the gas phase, (2) to explain which structures are more likely generated in the electrospray ion source, and (3) to determine which mechanisms are likely involved in their formation. Fragmentation pathways of the derived structures have also been investigated and rationalized to facilitate the interpretation of the data obtained under collisionally induced dissociation (CID) conditions.The present study confirms the assumption that ESI experiments with ions that are preformed in solution must be considered carefully. As a matter of fact, the study of ion formation mechanisms in the ion source is necessary to establish relationships between the ion structures in the condensed phase and the gas phase.